Our key objective of this research project will be to identify and characterize in molecular terms the primary cellular sites in muscle and adipose tissue which become defective due to the onset of obesity or diabetes, or both, and which contribute to the decreased responsiveness of these tissues to insulin action. Emphasis will be placed on the metabolic pathways for glucose in isolated fat cells as a model system. Techniques which monitor insulin-receptor interaction and new methods we have developed for the rapid direct measurement of transport processes in both brown and white fat cells will allow us to precisely quantitate receptors, the hexose transport system, and the coupling mechanism between these systems in cells from normal, obese, and diabetic animals. New information we have obtained using these techniques indicates that a major defect in at least one animal model of obesity occurs in intracellular enzyme activity which is involved in the metabolism of glucose. We plan to monitor the intracellular redox state of glutathione in fat cells (enzyme coupling assays) from normal and obese or diabetic animals since we have new evidence that the intracellular redox potential of this compound is intimately involved in regulating intracellular enzymes and transport system activity in fat cells. In addition we plan to study the effects of fatty acids on membrane transport processes in fat cells and to investigate the notion that their effects may be mediated through redox changes in glutathione, NADPH or other species secondary to oxidation of the fatty acids. These studies will be designed to further test the hypothesis that fatty acids may be a key factor in the insulin insensitivity seen in disease states by comparing their pattern of effects on these cellular parameters (insulin receptors, coupling system, transport system, intracellular enzyme activity) with the altered pattern seen in obesity or diabetes.